A novel near-infrared fluorescent probe (L) based on a 1,4-diethyl-1,2,3,4-tetrahydro-7H-pyrano[2,3-g]quinoxalin-7-one scaffold has been synthesized and characterized. Probe L displays highly selective and sensitive recognition to H₂S over various anions and biological thiols with a large Stokes shift (125 nm) in THF/H₂O (6/4, v/v, Tris–HCl 10 mM, pH = 7.4). This probe exhibits turn-on fluorescence for H₂S through HS⁻ induced thiolysis of dinitrophenyl ether. Confocal laser scanning micrographs of MCF-7 cells incubated with L confirm that L is cell-permeable and can successfully detect H₂S in living cells.